Objective: Meninges and extracranial tissues are innervated by small-diameter afferent nerve fibres that presumably fulfil nociceptive functions leading to headache and facial pain in humans. In rodents one or few small nerves ("spinosus nerve") directly arising from the trigeminal ganglion (TG) enter the cranial dura mater. Using retro- and anterograde tracing, we examined in which part of the TG the fibres of the spinosus nerve originate and to which tissues they project. Methods: The skull of adult Wistar rats was divided in the sagittal plane, the brain was removed and the spinosus nerve was cut near the TG. A crystal of the fluorescent tracer DiI was applied to the nerve stumps. The tissue was fixed in 4% PFA and incubated for 3–4 weeks at 37°C before it was examined with a fluorescence stereomicroscope. The TG was dissected, equilibrated in 30% sucrose buffer and cryocut. The dura mater was separated from the skull, immunohistochemically processed for CGRP and IB4 and examined as a whole mount. Results: Retrogradely traced nerve fibres were observed to arise from 150-200 somata of 10-55 mm in diameter, located preferably in the mandibular and maxillary region of the TG. Anterogradely traced fibers were seen in the dura mater following the middle meningeal artery and the superior sagittal sinus mainly in frontal direction. Part of the nerve fibres left the dura and passed into or through sutures of the skull, thereby innervating diploe, outer periost, dermis and splenus capitis, semispinalis and temporalis muscle. CGRP immunoreactive (-ir) nerve fibres were abundant while IB4-ir fibres were scarcely found. Conclusion: The rat cranial dura mater is richly innervated by nerve fibres of the spinosus nerve arising from neurons in mandibular and maxillary divisions of the TG. Surprisingly, a considerable proportion of partly CGRP immunopositive nerve fibres leave the dura innervating the skull or extracranial tissues. This presumably nociceptive innervation, if similar in humans, may contribute to the generation of referred pain and headaches.